1. Field of the Invention
Certain biological molecules, known as biological macromolecules, are characterized by high molecular masses (greater than 5000) and a tendency to auto-denaturation leading to a loss of biological activity or functional capacity (e.g. denaturation of proteins into peptides); these properties limit the separation methods applicable for industrial purification (precipitation, ultrafiltration and chromatography). Chromatography methods (selective adsorption) constitute the most specific and best adapted preparation techniques currently available for industrial production of biological macromolecules at high degrees of purity.
These methods comprise percolating a solution containing the biological macromolecules through a fixed bed of specific chromatographic resins suitable for leading to their selective adsorption. In the event of the desired macromolecule(s) being fixed to the resin, the elution of the latter with a solution of appropriate pH or ion charge allows it or them to be separated and collected in a purified and concentrated form. If the desired macromolecule remains in the treated solution (the other macromolecules being fixed to the resin) then the desired separation is obtained directly.
2. Description of Related Art
This fixed-bed chromatography technique and its results are well known in the bio-technology field and study of the following documents provides excellent examples:
"Method of Plasma Protein Fractionation", by J. M. CURLING, Academic Press, 1980 p. 149-160,
"Protein recovery by Ion Exchange", by D. T. JONES Bsc, Food Processing Industry, April 1975 P. 21, 23,
"New Methods of Valorization of Whey": by B. MIRABEL, Rhone-Poulenc Industries, "Information chimie" (Chemical News) n 175, March 1978 p. 105-109,
Patents FR No. 2.321.932 and 2.359.634 describing new ion-exchanging resins for protein separation.
However, these fixed-bed chromatography separation methods present some drawbacks, the most serious of which is the gradual clogging of the bed; this clogging is caused essentially by solid impurities in suspension in the solution and, often, by the precipitation of certain molecules of the solution itself.
The consequences are extremely serious from an industrial point of view. To maintain a constant flow of percolation over time the entry pressure of the solution must increase up to values in the order of 3 to 4 times greater than the nominal values which causes considerable technical difficulties and can even be unacceptable for certain applications. Furthermore, the cyclic cleaning of chromatographic resins is an extremely delicate operation in practice and generally necessitates cleaning in separate tanks, which prevents continuous operation of the equipment and imposes delicate and costly handling. It is to be noted that certain chromatographic resins, generally of a mineral nature, have fragile mediums which decompose during cleaning to give small particles which are subsequently likely to block certain zones of the bed and that other chromatographic resins, generally of an organic nature, cannot be cleaned when their meshes are blocked and so must be discarded. In certain cases, the blocking causes an integral saturation of the bed and cleaning is no longer possible. Another severe disadvantage of clogging is the change in fixation capacity of the resin bed after several cleanings: whatever precautions are taken a certain proportion of impurities remain fixed to the chromatographic resin and, little by little, its fixation capacity decreases until it becomes necessary to discard it.
These difficulties are well know to biological chromatography specialists and, in most cases, they necessitate a high degree of purification of the solutions before treatment. This purification greatly increases the cost of the method and limits its application for the production of high cost products (for example medicines ...)preventing its use in a great number of applications where the macromolecules produced have a lower value (treatment of certain natural products of a biological origin or by-products of industry especially the agricultural industry).
Another disadvantage of fixed-bed chromatography methods is the short life cycle of the resins which are rapidly destroyed by the constraints caused by their compression and the diverse intermediate cleaning and declogging treatments they undergo.
Currently, the problems mentioned above remain answered or unanswered at the price of a great increase in operating costs in the case of biological macromolecules; taking into account the economic importance of this type of treatment which affects not only the food sector but also the pharmaceutical sector, the veterinary sector, etc. . . . , several studies have been carried out to attempt to minimize the disadvantageous consequences summarized above in order to take maximum advantage of the performance of chromatographic methods for the separation of biological macromolecules. In this way certain writers have recommended the operation of a mobile compressed bed composed of a bed which is moved layer by layer by the removal of the resin at one extremity with its reinjection at the other extremity after cleaning (D. T. Jones BSc, "Protein Recovery by Ion Exchange"). However, no satisfactory solution for general application has so far been found, with certain serious disadvantages still existing such as for example, the need for cleaning, the deterioration of the fixation capacity and the wear of the chromatographic resins.
Besides, in other technical sectors, and in particular that of mineral separation, fluidized bed chromatography has long been carried out which avoids clogging and reduces the wear of the resins:
separation of metallic ions: "A Continuous Ion Exchange Column", by Turner and Church, Trans. Inst. Chem. Engrs, Vol 41, 1963 P. 283-288",
uranium extraction: "Assessment of Fluidized Bed Ion Exchange Equipment", Michael J. Slater, J. Appl. Chem. Biotechnol., 1975, vol. 25 p. 367-378".
These fluidized bed chromatography methods have been used for more than 20 years for the separation of molecules of small size (having good stability). However, biotechnicians have never tried to use them for biological macromolecule chromatography, because this technique has always appeared impossible to adapt to this type of treatment for various more or less objective reasons. Some relate, for specialists to the type of specific chromatographic resin for biological macromolecules (too small granulometry, density too close to that of water, unacceptable physical nature), which would be impossible to fluidize without drawing particles into the flux. Another reason concerns the kinetics of fixation of biological macromolecules which, mainly due to the large space between molecules, would be subject to a decrease of efficiency in a fluidized bed making the method inoperable. Another reservation concerns the infinitely small speed at which it would be necessary to draw the solution through a fluidized layer (not possible for industrial production).
So, already in 1980, the existing bias was sufficiently anchored in the mind of the specialists for an eminent biotechnician to write in an aforementioned publication "Methods of Plasma Protein Fractionation", J. M. Curling, p. 152":
"We have decided on a fixed-bed column rather than a fluidized bed column. Fluidization imposes a rising filtration flow and large rather than dense particles in order to maintain an equilibrium against the rising forces. But the available gels for protein fractioning are rarely denser than water and would give extremely diluted suspensions even at very weak flow speeds. In addition, because of the flow dispersion of macromolecules, the particle size and the inter-particle volumes should be as small as possible in order to increase the probability of the liquid element penetrating the porous internal volume. A fixed bed results in a minimal column volume and economizes on water consumption. All these arguments have not negligible financial consequences when choosing a general method. With all these requirements, it is evident that the exploitation of chromatography in the bio-industrial sector requires special apparatus having exceptional mechanical qualities. It must be possible to sterilize them when used in as compact a column as possible and to use as high a flow speed as possible for the daily treatment of several hundreds or thousands of liters of solution."
It must be noted that two earlier patents (GB patent No. 1.148.661 dated 09.05.66 and FR patent No. 2.105.032 dated 17/09/70) mention, in the case of one fixed-bed fluidized or open column chromatography for biological molecules, or for the other an ion exchange in a fluidized bed to purify hard water or uranium salts giving as a potential application the purification of biological substances. however, it is understandable that these patents were not able to prevent the bias, mentioned above, from forming. In fact, the former patent which envisages treatment in turbulent conditions, whether the bed be fixed fluidized or open, simply describes two examples of biological molecule chromatography both concerning small-sized molecules: codeine and atropin; besides this, in the general section, reference is made in this patent to a list of potential applications: hormones, vitamins, alkaloids, antibiotics, which are all small molecules apart from certain hormones. The second patent provides solely the examples of hard water purification to remove salts and of a uranium sale purification; this patent mentions in the general introduction, the application of the ion exchange process to biological substances and it is known that simple ion exchange processes are not applicable to small-sized molecules (contrary to the chromatography processes which involve selective chromatography resins of a specific type).
The present invention proposes an improvement of the current biological macromolecule chromatography separation technique in the biotechnology sector. It aims at the chromatography of molecules having a high molecular weight (greater than 5,000), such as proteins, enzymes, toxins, antibodies.
3. Objects
An essential objective of the present invention is to allow, in particular, the exploitation of chromatographic separation techniques avoiding the biggest drawback of these techniques, bed-clogging.
Another objective linked to the previous one is to reduce considerably the wear of the resin particles making up the bed.
Another objective is to authorize a continuous use of the separation techniques.
Another objective is to make biological macromolecule chromatographic separation feasible economically on an industrial scale for large numbers of particles of high-molecular weight, particularly to ensure the valorization of either agro-food industry protein by-products, or natural products such as milk, blood, plasma, or blood serum, or synthetic products.